AU710853B2 - Production of recombinant factor VIII in the presence of lipsome-like substances of mixed composition - Google Patents
Production of recombinant factor VIII in the presence of lipsome-like substances of mixed composition Download PDFInfo
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- AU710853B2 AU710853B2 AU52071/96A AU5207196A AU710853B2 AU 710853 B2 AU710853 B2 AU 710853B2 AU 52071/96 A AU52071/96 A AU 52071/96A AU 5207196 A AU5207196 A AU 5207196A AU 710853 B2 AU710853 B2 AU 710853B2
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- phosphatidylserine
- phosphatidylcholine
- liposome
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- 108010054218 Factor VIII Proteins 0.000 title claims abstract description 44
- 102000001690 Factor VIII Human genes 0.000 title claims abstract description 44
- 229960000301 factor viii Drugs 0.000 title claims abstract description 44
- 239000000126 substance Substances 0.000 title claims abstract description 36
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 20
- 239000000203 mixture Substances 0.000 title claims description 13
- TZCPCKNHXULUIY-RGULYWFUSA-N 1,2-distearoyl-sn-glycero-3-phosphoserine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCCCCCCCCCCCC TZCPCKNHXULUIY-RGULYWFUSA-N 0.000 claims abstract description 55
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 claims abstract description 55
- 210000004027 cell Anatomy 0.000 claims abstract description 24
- 230000014509 gene expression Effects 0.000 claims abstract description 22
- 150000002632 lipids Chemical class 0.000 claims abstract description 20
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 claims abstract description 14
- 150000008104 phosphatidylethanolamines Chemical class 0.000 claims abstract description 14
- 210000004962 mammalian cell Anatomy 0.000 claims abstract description 13
- 238000004113 cell culture Methods 0.000 claims abstract description 10
- 239000001963 growth medium Substances 0.000 claims abstract description 6
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 claims abstract description 5
- 102100026735 Coagulation factor VIII Human genes 0.000 claims abstract description 4
- SNKAWJBJQDLSFF-NVKMUCNASA-N 1,2-dioleoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC SNKAWJBJQDLSFF-NVKMUCNASA-N 0.000 claims abstract 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims description 30
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 claims description 30
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 claims description 24
- 235000012000 cholesterol Nutrition 0.000 claims description 15
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 claims description 12
- 235000021314 Palmitic acid Nutrition 0.000 claims description 12
- 235000020778 linoleic acid Nutrition 0.000 claims description 12
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 claims description 12
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 9
- 239000002609 medium Substances 0.000 claims description 6
- 239000006143 cell culture medium Substances 0.000 claims description 5
- 239000012634 fragment Substances 0.000 claims description 5
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 3
- 229920001184 polypeptide Polymers 0.000 claims description 2
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 2
- 239000004417 polycarbonate Substances 0.000 description 23
- 150000003904 phospholipids Chemical class 0.000 description 12
- 230000000694 effects Effects 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 230000007774 longterm Effects 0.000 description 3
- 239000012679 serum free medium Substances 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 108010089996 B-domain-deleted factor VIII Proteins 0.000 description 2
- 101100209914 Mus musculus Vill gene Proteins 0.000 description 2
- 210000001766 X chromosome Anatomy 0.000 description 2
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 2
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- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 238000010899 nucleation Methods 0.000 description 2
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- 102000004169 proteins and genes Human genes 0.000 description 2
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- 108010017384 Blood Proteins Proteins 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
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- 206010053567 Coagulopathies Diseases 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 201000003542 Factor VIII deficiency Diseases 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 208000009292 Hemophilia A Diseases 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 102100036881 Inositol-3-phosphate synthase 1 Human genes 0.000 description 1
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- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 1
- 102000007330 LDL Lipoproteins Human genes 0.000 description 1
- 108010007622 LDL Lipoproteins Proteins 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 102000004338 Transferrin Human genes 0.000 description 1
- 108090000901 Transferrin Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 239000010441 alabaster Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 208000034158 bleeding Diseases 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 1
- 125000000837 carbohydrate group Chemical group 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000035602 clotting Effects 0.000 description 1
- 230000004186 co-expression Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 239000013256 coordination polymer Substances 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
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- 230000007717 exclusion Effects 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 235000021588 free fatty acids Nutrition 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000012913 medium supplement Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 239000007758 minimum essential medium Substances 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000013587 production medium Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000012807 shake-flask culturing Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 239000012581 transferrin Substances 0.000 description 1
- 108010047303 von Willebrand Factor Proteins 0.000 description 1
- 102100036537 von Willebrand factor Human genes 0.000 description 1
- 229960001134 von willebrand factor Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/745—Blood coagulation or fibrinolysis factors
- C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
- C12N5/0037—Serum-free medium, which may still contain naturally-sourced components
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/36—Lipids
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- Gastroenterology & Hepatology (AREA)
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- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract
Recombinant Factor VIII expression in a mammalian cell culture can be increased by including a novel liposome-like substance in the culture medium. The liposome-like substance comprises at least two (preferably at least three) different lipids in defined molar ratios. In a preferred embodiment, the addition of a liposome-like substance comprised of dioleoyl phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine in a molar ratio of 4:1:1 to the culture medium of GS-MDR cells resulted in an increase in FVIII production by a factor greater than five.
Description
Patent MSB-7226-CIP BACKGROUND OF THE INVENTION Eiedd This disclosure is concerned generally with the production of recombinant Factor VIII in a mammalian cell expression system. Specifically, the disclosure relates to the addition of a liposome-like substance containing lipids in defined ratios to the mammalian cell culture medium to increase yields of recombinant Factor VIII.
Background Factor VIII is a plasma protein required for normal hemostasis, or clotting of the blood. Functional Factor VIII is lacking in individuals with hemophilia A because of a mutation in the gene encoding this protein, which is located in the X-chromosome. To control bleeding episodes, hemophiliacs must be treated with Factor VIII, which historically has been isolated from human blood plasma.
The human Factor VIII gene encompasses 186,000 base pairs and constitutes 0.1 of the entire X-chromosome, making it among the largest genes known The transcription product of this gene, which is derived from 26 exons, is a messenger RNA molecule of -9000 bases in length, coding for a large protein of 2351 amino acids. Structural studies of Factor VIII indicate that it is a glycoprotein, containing a significant number of carbohydrate residues. The cDNA coding for Factor VIII has been cloned and stably expressed in baby hamster kidney cells (BHK-21) and Chinese hamster ovary cells The availability of these high producing cell clones has made large-scale production of recombinant Factor VIII (rFVIII) feasible. Two significant challenges in the commercial production of rFVIII are the development of a serumfree medium that will support high density cultures and stabilize rFVIII, and (ii) an efficient purification scheme that will yield high purity rFVIII.
Previously it has been demonstrated that the addition of bovine lipoprotein or human low density lipoprotein to serumfree cultures significantly improve the productivity of recombinant BHK-21 and human embryonic kidney (293S) cells expressing rFVIII The co-expression of vonWillebrand factor and the addition of phospholipids to serumfree medium have been shown to be effective in enhancing the stability of rFVIII produced by rFVIII expressing CHO cells I have found that certain liposome-like substances comprising at least two (preferably at least three) lipids can be used as culture supplements in the serumfree production of rFVIII.
Contrary to the prior art I have observed that certain liposome-like sustances comprised of lipids such as phosphatidylcholine(PC), phosphatidylethanolamine or phosphatidylserine (PS) alone have no effect on rFVIII expression in BHK-21 and 293S cells. However, liposome-like substances comprising combinations of different lipids, such as cholesterol, fatty acids such as linoleic acid and palmitic acid, PC, PE, and PS, at certain ratios were found to have a significant enhancing effect on rFVIII expression in BHK-21 and 293S cells. A serumfree production medium for long term production of rFVIII was developed from these new findings.
SUMMARY OF THE INVENTION Thus a method and medium has been found for substantially increasing the productivity of a mammalian cell expression system producing recombinant Factor VIII. The essential step of the method consists of the addition of a liposome-like substance to the cell growth medium of the expression system.
i According to an aspect of the invention there is provided a method for increasing the production level of recombinant factor VIII in a mammalian cell culture expression system by at least fourfold, comprising the step of adding to the culture system fixed molar ratios of a mixture of synthetic lipids in the form of a liposome-like substance selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine in a molar ratio of about 4:1:1, phosphatidylcholine, phosphatidylserine and cholesterol in molar ratio of about 8:1:1 and phosphatidylcholine, phosphatidylserine, palmitic acid and linoleic acid in a molar ratio of about 7:3:0.5:0.5 under conditions sufficient to assure the fourfold increase in productivity.
According to another aspect of the invention there is provided a cell culture medium containing fixed molar ratios of a mixture of synthetic lipids in the form of liposome-like substance, wherein said liposome-like substance is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine in a molar ratio of about 4:1:1, phosphatidylcholine, phosphatidylserine and cholesterol in a molar ratio of about 8:1:1 and phosphatidylcholine, phosphatidylserine, palmitic acid and linoleic acid in a molar ratio of about 7:3:0.5:0.5 sufficient to assure a fourfold increase in factor VIII expression in a mammalian cell culture system.
According to yet another aspect of the invention there is provided a method for increasing the production level of recombinant factor VIII in a mammalian cell culture expression system by at least threefold, comprising the step of adding to the culture system fixed molar ratios of a mixture of synthetic lipids in the form of a liposome-like substance selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine in a molar ratio of about 4:1:1, phosphatidylcholine, phosphatidylserine and cholesterol in molar ratio of about 8:1:1 and phosphatidylcholine, phosphatidylserine, palmitic acid and linoleic acid in a molar ratio of about 7:3:0.5:0.5 under conditions sufficient to assure the threefold increase in productivity, wherien the recombinant factor VIII is truncated and the liposome-like substance is comprised of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine in a molar ratio of about 8:1:1.
Throughout this specification and the claims which follow, unless the text requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
a a Patent MSB-7 22fi-CLe different lipids in fixed molar ratios. Molar ratios should be understood to be approximate.
This invention is illustrated in the following examples, which set forth typical procedures and cell culture media for production (preferably continuous production) of rFVIII using the liposome-like substances to deliver lipid supplements to recombinant cells expressing high levels of rFVIII.
SPECIFIC EMBODIMENTS Preparation of liDosome-like substances All synthetic phospholipids were obtained from Avanti Polar Lipids, Inc. (Alabaster, Alabama). Phospholipids were delivered as 0.1 p liposome-like substances. Lyophilized phospholipids were reconstituted in 50 mM Tris-150 mM NaCI (pH 7.4) and extruded through a 0.1 polycarbonate membrane with the aid of a hand-held device LiposoFast S° (Avestin, Inc., Ottawa, Canada). The sized liposome-like substances were then S. filtered with a 0.2 p filter and added aseptically to culture medium.
Phosphatidylethanolamine phosphatidylcholine and phosphatidylserine (PS) were examined either as single components or mixtures. Other lipids such as cholesterol and free fatty acids were also incorporated into the liposome-like substances.
JO
Patent MSB-7226f-Cp EXAMPLE
I
Effect of various lipid mixtures on the expression of rFVl in 293S cell (recombinant 293S cells expressing high levels of Factor VIII) cells were maintained as serumfree cultures in shake flasks using a serumfree medium (Dulbecco's minimum essential medium and F12 at a ratio of 1:1, obtained from Life Technologies, Bethesda, MD) supplemented with insulin (10 /g/ml) and transferrin (25 pg/ml). Long term evaluation was done in shake flasks with an initial seeding density of 3 x 106 cells/ml. Complete medium exchanges were done at 24-hour intervals where cells were spun, washed and reseeded at 3 x 106 cells/ml. A typical shaker culture contains 25 50 ml of cells. Factor VIII activity was determined by Coatest VIII (Kabi Pharmacia, Franklin, Ohio), a S chromogenic assay, according to manufacturer's instructions.
o. The initial screening of phospholipids was done using 24-hour plate cultures.
S After determining the optimal ratio of various phospholipids, the study was then confirmed in shake flasks over a period of 10 14 days. As shown in Table 1, while PC and PE alone had no effect on Factor VIII expression, PS alone was found to be inhibitory. By combining PC, PS, and PE at various ratios, significant i ncreases in Factor VIll expression were observed. The highest productivity was observed in cells supplemented with PC:PE:PS PC:PS:cholesterol and PC:PS:palmitic acid:linoleic acid The optimal concentration of phospholipids was found to be 30 pg/ml. The optimal length of the acyl side chain of various phospholipids was determined to be C18. All optimization studies were subsequently done with dioleoyl phospholipids.
Patent MSB-7226-CIP EXAMPLE II Expression of factor VIII in continuous culture I measured the effect of various liposome-like substances on the production of factor VIII in long term shake flask cultures with PC:PE:PS PC:PS: palmitic acid:linoleic acid and PC:PS:cholesterol The culture conditions were done as described in Example I. The concentration of the liposome-like substances was at 30 /ug/ml.
Complete medium exchange was done at 24-hour intervals. Results are shown in Table 2.
EXAMPLE III 0 Expression of Factor VIII in continuous cultures The effect of various liposomes on the production of truncated Factor VIII (deletion of all or part of the B domain of Factor VIII) was examined in recombinant 293S cells expressing high levels of a B-domain-deleted Factor VIII with the following sequence (SEQ ID NO. 1) joining the 90-kD and fragments of Factor VIll: 0* 40*0 S 90-kD--SFSQNPPVLKRHQR--80kD 0* (Amino acid abbreviations are as given in Ref. incorporated herein by Sc reference.) This truncated Factor VIII is essentially as described in Ref. incorporated herein by reference.
Patent MISB-7226-CP The culture conditions were done in 12-well plates with an initial seeding density of 2 x 105 cells per well in DMEM/F12 supplemented with 5% fetal bovine serum. After confluency was reached the cells were washed with PBS and fed with the serumfree production as described in Example I. Results are shown in Table 3, where at least a threefold increase in productivity over the saline control is shown. The highest productivites were observed in cells supplemented with dioleoyl PC/PE/PS and dioleoyl PC/PS/cholesterol
CONCLUSION
We have demonstrated that lipid mixtures, when delivered in the form of S* liposome-like substances, significantly enhance the production of Factor VIII (in both full length and truncated forms) in recombinant cells. These liposome-like substances can be used as medium supplements to support production of Factor VIII, preferably continuous production of Factor VIII. As used herein, the term Factor VIII is intended to include all variants or truncated forms of Factor VIII having Factor VIII activity.
The above examples are intended to illustrate the invention and it is thought variations will occur to those skilled in the art. Accordingly, it is intended that Sthe scope of the invention should be limited only by the claims below.
0 0 Patent Effect of phospholipid on the expression of Factor VIII in GS-MDR cells Table 1.
IFPhospholipids FVIII Titer (U/mI) Dioleoyl PG/PS 2.8 Dioleoyl Pc/Ps Dioleoyl PC/PS 1.4 Dioleoyl PC/PSlpalmitic/Iinoleic acid 3.4 Dioleoyl PC/PEIPS Dioleoyl PC/PE/PS 1.7 DioleoylPC/PE/PS (16:1:2) 2.1 Dioleoyl PC 0.65 -DioleoyliPE-- 0.55 Dioleoyl PS [0.1 Saline 0.60 0* 0 0 0 OS.6 00 S 0 5550 0 50 S 0.
S S 000 5 Patent MSB-7226CIP Table 2. Production of Factor VIII in continuous cultures of GS-10 cells Days titer (U/mi) PC/PEIPS PC/PS/pm/In PC/PS/Cholesterol Medium only (8:1 12.44 2.51 2.41 0.35 2 2.78 2.89 2.72 0.4 3 2.46 2.77 2.69 0.44 4 2.82 2.8 2.7 0.48 2.88 2.95 2.99 0.42 6 2.9 3.1 2.95 0.41 7 3.21 3.18 3.12 0.54 8 3.18 3.22 3.06 0.55 9 3.02 3.16 3.14 0.58 10 3.34 3.38 3.27 0.53 11 2.97 3.15 3.19 0.49 12 3.12 2.95 2.98 0.52 13 3.02 3.12 2.71 0.54 14 2.89 3.16 3.22 0.56 15 -3.22, 3.38 3.34 0.51
S
0**SS*
S
S.
5S5
S~~S
S
55 5 S S *5 05
S
S*S S pm =palmitic acid In linoleic acid 55 S S 5O5* 5@OS
S
5* 0 55 S S See S 05 5 0
S
Patent MSB-226-CIPE Table 3. Effect of phospholipid on the expression of a B-domain deleted Factor VIII variant in 293S cells.
Phospholipids JFVIII Titer (U/mI) Dioleoyl Pc/PS 2.80 Dioleoyl Pc/PS 0.85 Dioleoyl PC/PS/Cholesterol Dioleoyl PC 1 .0 Dioleoyl PC/PE/IPS 1: 1) 3.04 Saline ]0.9 0 0000 0* 0*
S@
es 0 0 I1.
2.
3.
.4.
8..
REFERENCES
Gitschier et at. 1 984 Nature 312 326 329 Wood et al. 1984 Nature 312 :330 337 Toole et al. 1984 Nature 312 :342 347 Kaufman et al. 1989 Mol. Cell Biol. 9 1233 -1242 Chan et al. 1991 In Vitro 27 :121 Kaufman et al. 1 0/1 993 U.S. Patent 5,250,421 Scholz et al. 5/1993 U.S. Patent 5,210,075 Almstedt et al. 6/1991 WO 91/09122 Patent MSB-7226-CIP SEQUENCE LISTING 1) GENERAL INFORMATION: APPLICANTS: Chan, Sham-Yuen (ii) TITLE OF INVENTION: Production of Recombinant Factor VIII in the Presence of Liposome-like Substances of Mixed Composition (iii) NUMBER OF SEQUENCES: 1 (iv) CORRESPONDENCE
ADDRESS:
ADDRESSEE: Bayer Corporation STREET: 800 Dwight Way P. O. Box 1986 CITY: Berkeley STATE: California COUNTRY:
USA
ZIP: 94701-1986 COMPUTER READABLE FORM: MEDIUM TYPE: Diskette, 3.50 inch, 1.44Mb Storage COMPUTER: IBM OPERATING SYSTEM: DOS SOFTWARE: WordPerfect 6.1 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING
DATE:
CLASSIFICATION:
(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: 08/434,900 FILING DATE: May 4, 1995 (viii) ATTORNEY/AGENT
INFORMATION:
A) NAME: Giblin, James A.
REGISTRATION NUMBER: 25772 REFERENCE/DOCKET NUMBER: MSB-7226CIP ix) TELECOMMUNICATION
INFORMATION:
TELEPHONE: (510)705-7910 TELEFAX: (510)705-7904 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 14 TYPE: amino acid STRANDEDNESS: single strand TOPOLOGY: linear (ii) MOLECULE TYPE: DESCRIPTION: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: Ser-Phe-Ser-Gln-Asn-Pro-Pro-Val-Leu-Lys-Arg-His-Gln-Arg 1 5 The claims defining the invention are as follows: 1. A method for increasing the production level of recombinant factor VIII in a mammalian cell culture expression system by at least fourfold, comprising the step of adding to the culture system fixed molar ratios of a mixture of synthetic lipids in the form of a liposome-like substance selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine in a molar ratio of about 4:1:1, phosphatidylcholine, phosphatidylserine and cholesterol in molar ratio of about 8:1:1 and phosphatidylcholine, phosphatidylserine, palmitic acid and linoleic acid in a molar ratio of about 7:3:0.5:0.5 under conditions sufficient to assure the fourfold increase in productivity.
2. A method for increasing the production level of recombinant factor VIII in a mammalian cell culture expression system by at least fourfold, comprising the step of adding to the culture system fixed molar ratios of a mixture of synthetic lipids in the form of a liposome-like substance selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine in a molar ratio of about 4:1:1, phosphatidylcholine, phosphatidylserine and cholesterol in molar ratio of about 8:1:1 and phosphatidylcholine, phosphatidylserine, palmitic acid and linoleic acid in a molar ratio of about 7:3:0.5:0.5 under conditions sufficient to assure the fourfold increase in productivity, wherein the recombinant factor VIII is truncated and the liposome-like substance is comprised of phosphatidylcholine, phosphatylethanolamine and phosphatidylserine in a molar a o• oratio of about 8:1:1.
3. The method of claim 2, wherein the recombinant factor VIII has a 90kD fragment 9 and an 80kD fragment which are linked by a polypeptide having a sequence Ser- Phe-Ser-Gln-Asn-Pro-Pro-Val-Leu-Lys-Arg-His-Gln-Arg.
Claims (3)
- 4. A cell culture medium containing fixed molar ratios of a mixture of synthetic lipids in the form of liposome-like substance, wherein said liposome-like substance is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine in a molar ratio of about 4:1:1, phosphatidylcholine, phosphatidylserine and cholesterol in a molar ratio of about 8:1:1 and phosphatidylcholine, phosphatidylserine, palmitic acid and linoleic acid in a molar ratio of about 7:3:0.5:0.5 sufficient to assure a fourfold increase in factor VIII expression in a mammalian cell culture system. A cell culture medium containing fixed molar ratios of a mixture of synthetic lipids in the form of liposome-like substance, wherein said liposome-like substance is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine in a molar ratio of about 4:1:1, phosphatidylcholine, phosphatidylserine and cholesterol in a molar ratio of about 8:1:1 and phosphatidylcholine, phosphatidylserine, palmitic acid and linoleic acid in a molar ratio of about 7:3:0.5:0.5 sufficient to assure a fourfold increase in factor VIII expression in a mammalian cell culture system, wherein the recombinant factor VIII is truncated and the liposome-like substance is comprised of phosphatidylcholine, phosphatylethanolamine and phosphatidylserine in a molar ratio of about 8:1:1. C
- 6. The medium of claim 5, wherein the truncated factor VIII has a 90 kD fragment and an 80kD fragment which are linked by a polypeptide having a sequence Ser-Phe- Ser-Gln-Asn-Pro-Pro-Val-Leu-Lys-Arg-His-Gln-Arg. -13-
- 7. A method for increasing the production level of recombinant factor VIII in a mammalian cell culture expression system by at least threefold, comprising the step of adding to the culture system fixed molar ratios of a mixture of synthetic lipids in the form of a liposome-like substance selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine in a molar ratio of about 4:1:1, phosphatidylcholine, phosphatidylserine and cholesterol in molar ratio of about 8:1:1 and phosphatidylcholine, phosphatidylserine, palmitic acid and linoleic acid in a molar ratio of about 7:3:0.5:0.5 under conditions sufficient to assure the threefold increase in productivity, wherien the recombinant factor VIII is truncated and the liposome-like substance is comprised of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine in a molar ratio of about 8:1:1. DATED this 4 th day of August 1999 BAYER CORPORATION By its Patent Attorneys DAVIES COLLISON CAVE a a S °000 Patent MSB-7226-Cp ABSTRACT Recombinant Factor VIII expression in a mammalian cell culture can be increased by including a novel liposome-like substance in the culture medium. The liposome-like substance comprises at least two (preferably at least three) different lipids in defined molar ratios. In a preferred embodiment, the addition of a liposome-like substance comprised of dioleoyl phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine in a molar ratio of 4:1:1 to the culture medium of GS-MDR cells resulted in an increase in FVIII production by a factor greater than five. 4*04 X*
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/434900 | 1995-05-04 | ||
| US08/434,900 US5679549A (en) | 1995-05-04 | 1995-05-04 | Production of recombinant factor VIII in the presence of liposome-like substances of mixed composition |
| US08/634001 | 1996-04-17 | ||
| US08/634,001 US5952198A (en) | 1995-05-04 | 1996-04-17 | Production of recombinant Factor VIII in the presence of liposome-like substances of mixed composition |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| AU5207196A AU5207196A (en) | 1996-11-28 |
| AU710853B2 true AU710853B2 (en) | 1999-09-30 |
Family
ID=27030345
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU52071/96A Ceased AU710853B2 (en) | 1995-05-04 | 1996-05-03 | Production of recombinant factor VIII in the presence of lipsome-like substances of mixed composition |
Country Status (7)
| Country | Link |
|---|---|
| US (1) | US5952198A (en) |
| EP (1) | EP0745672B1 (en) |
| JP (1) | JP3996655B2 (en) |
| AT (1) | ATE311447T1 (en) |
| AU (1) | AU710853B2 (en) |
| CA (1) | CA2175628C (en) |
| DE (1) | DE69635504T2 (en) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6200560B1 (en) * | 1998-10-20 | 2001-03-13 | Avigen, Inc. | Adeno-associated virus vectors for expression of factor VIII by target cells |
| US6358703B1 (en) | 1998-12-10 | 2002-03-19 | Bayer Corporation | Expression system for factor VIII |
| WO2002061036A2 (en) * | 2000-11-30 | 2002-08-08 | The Research Foundation Of State University Of New York | Method of complexing a protein by the use of a dispersed system and proteins thereof |
| US7625584B2 (en) * | 2000-11-30 | 2009-12-01 | The Research Foundation Of State University Of New York | Method of complexing a protein by the use of a dispersed system and proteins thereof |
| AU2002239607A1 (en) * | 2000-11-30 | 2002-06-11 | Baxter Healthcare Corporation | Ahf associated dispersion system and method for preparation |
| US8110218B2 (en) * | 2000-11-30 | 2012-02-07 | The Research Foundation Of State University Of New York | Compositions and methods for less immunogenic protein-lipid complexes |
| US20050221414A1 (en) | 2004-03-31 | 2005-10-06 | Katalin Varadi | Kit for measuring the thrombin generation in a sample of a patient's blood or plasma |
| EP1988101A1 (en) * | 2007-05-04 | 2008-11-05 | Novo Nordisk A/S | Improvement of factor VIII polypeptide titers in cell cultures |
| WO2013160005A1 (en) * | 2012-04-24 | 2013-10-31 | Novo Nordisk A/S | Pharmaceutical composition suitable for treatment of haemophilia |
| KR20160125352A (en) * | 2013-12-20 | 2016-10-31 | 이센셜 파마수티컬스 엘엘씨 | Media for cell culture |
| EA202190827A1 (en) * | 2015-11-13 | 2021-11-30 | Баксалта Инкорпорейтед | VIRAL VECTORS CODING RECOMBINANT FVIII VARIANTS WITH INCREASED EXPRESSION FOR GENE THERAPY OF HEMOPHILIA A |
| JP2018023356A (en) * | 2016-08-04 | 2018-02-15 | 三洋化成工業株式会社 | Method for producing useful substance |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1987004187A1 (en) * | 1986-01-03 | 1987-07-16 | Genetics Institute, Inc. | METHOD FOR PRODUCING FACTOR VIII:c-TYPE PROTEINS |
| EP0254076A1 (en) * | 1986-07-11 | 1988-01-27 | Miles Inc. | Improved recombinant protein production |
| US5250421A (en) * | 1986-01-03 | 1993-10-05 | Genetics Institute, Inc. | Method for producing factor VIII:C-type proteins |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5198349A (en) * | 1986-01-03 | 1993-03-30 | Genetics Institute, Inc. | Method for producing factor VIII:C and analogs |
-
1996
- 1996-04-17 US US08/634,001 patent/US5952198A/en not_active Expired - Lifetime
- 1996-04-25 EP EP19960106482 patent/EP0745672B1/en not_active Expired - Lifetime
- 1996-04-25 AT AT96106482T patent/ATE311447T1/en not_active IP Right Cessation
- 1996-04-25 DE DE1996635504 patent/DE69635504T2/en not_active Expired - Lifetime
- 1996-05-01 JP JP13254696A patent/JP3996655B2/en not_active Expired - Fee Related
- 1996-05-02 CA CA 2175628 patent/CA2175628C/en not_active Expired - Fee Related
- 1996-05-03 AU AU52071/96A patent/AU710853B2/en not_active Ceased
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1987004187A1 (en) * | 1986-01-03 | 1987-07-16 | Genetics Institute, Inc. | METHOD FOR PRODUCING FACTOR VIII:c-TYPE PROTEINS |
| US5250421A (en) * | 1986-01-03 | 1993-10-05 | Genetics Institute, Inc. | Method for producing factor VIII:C-type proteins |
| EP0254076A1 (en) * | 1986-07-11 | 1988-01-27 | Miles Inc. | Improved recombinant protein production |
Also Published As
| Publication number | Publication date |
|---|---|
| DE69635504D1 (en) | 2006-01-05 |
| EP0745672A3 (en) | 1998-05-20 |
| AU5207196A (en) | 1996-11-28 |
| JP3996655B2 (en) | 2007-10-24 |
| CA2175628A1 (en) | 1996-11-05 |
| JPH0998796A (en) | 1997-04-15 |
| CA2175628C (en) | 2007-02-20 |
| EP0745672B1 (en) | 2005-11-30 |
| US5952198A (en) | 1999-09-14 |
| EP0745672A2 (en) | 1996-12-04 |
| DE69635504T2 (en) | 2006-06-14 |
| ATE311447T1 (en) | 2005-12-15 |
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